Effects of treponema denticola on an oral epithelial cell model

Perïodontal diseases are destructive inflammatory conditions of the tissues supporting the tooth. The inflanmatory responses are induced by mixed bacteriai infection leading to eventual destruction of the periodontal tissue. Oral spirochetes of the genus Treponerna are one of the bacterial groups that are closely related to these diseases. This study investigated the interaction of a periodontal pathogen, T. denticola, its whole cells and outer membrane (OM) proteins with cultured eukaryotic cells. Cultured porcine periodontal ligament epithelial cells (PLE, a junctional epithelium model), and various ce11 lines were challenged with T. denticola ATCC 35405 whole cells or with the major OM protein (Msp) complex, gel purified Msp, and chymotrypsin-like proteinase (CTLP). Msp complex was isolated fiom whole T. denticdu by detergent extraction, autoproteolysis, ultrd~ltrationa nd detergent removai. The location of the Msp and CTLP on T. denticola OM was studied. Hexagonal array was observable on negativestained T. dentkola OM under electron microscope where Msp probably constituted the main building block of the regular pattern together with the previousiy reported OM located CTLP. Native i? denticola Msp is an oligomer containing 53 kDa protein subunits which had porin activity in an artificial lipid bilayer. CTLP was previously characterized. Microscopic and modified ELISA assays showed that spirochetes adhered to PLE cells whether prefixed with glutaraidhyde (fixed PLE-FPLE) or not. Pre-treatrnent of T. denticda with the protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone or phenylrnethanesulfonyl fluoride blocked this binding and chymotrypsin-like activity. T denficola intact or sonicated whole cells were cytotoxic to PLE cells and to several ce11 lines. microculture tetrazolium assay or by quantiQing (LDH assay). T. denticola induced PLE ceIIda ii T. denticoh cytotoxicity was measured by lactate dehydrogenase release upon ce11 lysis changes were observed by light microscopy, image aaalysis and electron microscopy. These included: transient ce11 size increase of nondetached PLE cells leading to confluency maintenance, membrane disruption, vacuolation, loss of cell contacts, loss of ce11 size control, cytoskeletal rearrangement, and apoptosis of PLE cells. Msp complex, Msp and CTLP could attach to PLEFPLE and were found to be cytotoxic to PLE cells. Adherence of Msp was partiaily blocked by specific antibodies. Adherence of CTLP was paaially blocked by serine protease inhibiton and was M e r inhibited by specific antibodies. Cytotoxicity of Msp and CTLP was inhibited by the same treatments that inhibited adherence. Standard patch clamp recording methods were used to study the effects of Msp complex on HeLa cells. Msp bound to several HeLa ce11 proteins, including a 65 kDa surface protein and a 96 kDa cytoplasmic protein. The Msp complex depolarized and increased the conductance of the HeLa ce11 membrane in a manner that was not strongly selective for Na', K', ~a",a nd CI- ions. Cellattached patches of HeLa cell membrane exposed to Msp complex exhibited short-lived channels with a dope conductance of 0.4 nS in physiologically normal saline. These studies show that i? denlicola ATCC 35405 and its major OM protein elements, namely Msp and CTLP, could attach to a ce11 mode1 that resembles junctional epithelium and could produce cytopathic and cytotoxic effects. It was hypothesized that the strong proteolytic activities of CTLP and pore- Forming activity of the Msp complex were responsible for the cytopathic and cytotoxic effects of the putative periodontopathogen T. denticola on PLE cells.published_or_final_versionDoctoralDoctor of Philosoph